U.S. patent number 8,096,484 [Application Number 12/377,479] was granted by the patent office on 2012-01-17 for method for the production of dry free-flowing hydrophobin preparations.
This patent grant is currently assigned to BASF SE. Invention is credited to Tillman Faust, Marvin Karos, Ulrike Richter, Michael Schonherr, Thomas Subkowski.
United States Patent |
8,096,484 |
Schonherr , et al. |
January 17, 2012 |
**Please see images for:
( Certificate of Correction ) ** |
Method for the production of dry free-flowing hydrophobin
preparations
Abstract
Method for the production of dry, free-flowing, stable
hydrophobin preparations by spraying and drying aqueous hydrophobin
solutions, if appropriate comprising additives in a spray
device.
Inventors: |
Schonherr; Michael
(Frankenthal, DE), Faust; Tillman (Weisenheim,
DE), Richter; Ulrike (Jersey City, NJ), Subkowski;
Thomas (Ladenburg, DE), Karos; Marvin
(Schwetzingen, DE) |
Assignee: |
BASF SE (DE)
|
Family
ID: |
38516118 |
Appl.
No.: |
12/377,479 |
Filed: |
August 6, 2007 |
PCT
Filed: |
August 06, 2007 |
PCT No.: |
PCT/EP2007/058103 |
371(c)(1),(2),(4) Date: |
February 13, 2009 |
PCT
Pub. No.: |
WO2008/019965 |
PCT
Pub. Date: |
February 21, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100317833 A1 |
Dec 16, 2010 |
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Foreign Application Priority Data
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Aug 15, 2006 [EP] |
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06118947 |
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Current U.S.
Class: |
239/9 |
Current CPC
Class: |
F26B
3/12 (20130101); F26B 3/08 (20130101); A23J
3/20 (20130101); A23L 31/00 (20160801) |
Current International
Class: |
A62C
5/02 (20060101) |
Field of
Search: |
;530/350 ;427/372.2
;239/9 |
References Cited
[Referenced By]
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|
Primary Examiner: Monshipouri; Maryam
Attorney, Agent or Firm: Connolly Bove Lodge & Hutz
LLP
Claims
The invention claimed is:
1. A method for producing a dry, free-flowing, stable hydrophobin
preparation comprising spraying and drying a hydrophobin solution
and, optionally, an additive, in a spray device, wherein the
spraying takes place at an inlet air temperature of 120.degree. C.
to 200.degree. C.
2. The method of claim 1, wherein the hydrophobin solution is
sprayed with co-use of from 5% to 200% by weight, based on the
hydrophobin solids content of the solution, of an organic or
inorganic spray auxiliary, wherein particles laden with the spray
auxiliary are dried.
3. The method of claim 1, wherein the hydrophobin preparation is in
the form of granules, and wherein a fluidized bed is used in
addition to the spray device.
4. The method of claim 2, wherein the organic or inorganic spray
auxiliary further comprises one or more sugar alcohols, celluloses,
or starch.
5. The method of claim 2, wherein the organic or inorganic spray
auxiliary comprises mannitol.
6. The method of claim 1, wherein the spraying takes place
alternatively or additionally at an outlet air temperature of from
50.degree. C. to 120.degree. C.
Description
RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C.
.sctn.371) of PCT/EP2007/058103, filed Aug. 6, 2007, which claims
benefit of European Application No. 06118947.8, filed Aug. 15,
2006.
The invention relates to an improved method for the production of
dry, free-flowing, stable hydrophobin preparations by spraying an
aqueous hydrophobin solution in a spraying device. It further
relates to hydrophobin preparations produced by this method which
comprise these hydrophobin preparations.
The isolation of hydrophobins usually occurs as aqueous solution.
In this form, the hydrophobins lose their activity or are
microbially decomposed and can only be stored and transported with
great expenditure. It is therefore desirable to produce dry
preparations of hydrophobins which comprise the hydrophobin in
concentrated form with the lowest possible loss of its specific
effect. Furthermore, these preparations should consist of particles
with well developed surface in a particle size of from 50 to 600
.mu.m, so that, in the further-processing industry, a homogeneous
mixture of these products with other substances or a good
applicability is ensured.
There are various spray methods for removing water from
enzyme-containing aqueous media.
In the U.S. Pat. No. 4,617,272, enzyme-containing media are sprayed
onto inert particles heated in a fluidized bed.
Polyolefins, polycarbonates, polymethyl methacrylates or
polystyrene are specified as suitable inert particles.
One disadvantage of this method is that the dry enzyme powders
produced by means of these inert particles cannot be used in the
food and animal feed industry.
According to another method, which is described in the Economic
Patent DD 263 790, milk curdling protease products are produced by
spraying aqueous protease solutions onto carrier substances located
in a fluidized-bed granulator. Skimmed milk powder and/or
dextrin-containing substances are described as carrier
substances.
Although the resulting products have good enzyme stability and
pourability and the carrier substances used are physiologically
compatible, this method has the disadvantage that up to 10 times
the amount of carrier substances, based on enzyme solid, has to be
used.
EP 522269 describes a method for the production of dry,
free-flowing, stable enzyme preparations by spraying aqueous enzyme
solutions, if appropriate comprising additives, in a spray device,
wherein the enzyme solution is sprayed with co-use of from 5 to 60%
by weight, based on the enzyme solids content of the solution, of a
spray auxiliary consisting of hydrophobic silica and/or a metal
salt of a higher fatty acid at 0 to 50.degree. C., and the
particles laden with spray auxiliaries obtained in this way are
dried.
It was therefore an object of the present invention to propose a
method which allows aqueous hydrophobin solutions to be converted
into dry, stable hydrophobin preparations which can be used in the
further-processing industry.
It has now been found that the method defined at the start leads to
particularly highly suitable hydrophobin preparations when the
aqueous hydrophobin solution, if appropriate comprising additives,
is sprayed with co-use of from 5 to 200% by weight, based on the
hydrophobin solids content of the solution, of a spray auxiliary
consisting of one or more sugar alcohols, and the particles laden
with spray auxiliaries obtained in this way are dried.
Hydrophobins are small proteins of about 100 AA which are
characteristic of filamentous fungi and do not occur in other
organisms. Recently, hydrophobin-like proteins were discovered in
Streptomyces coelicolor, which are referred to as "chaplins" and
likewise have high surface-active properties. At water/air
interfaces, chaplins can assemble to give amyloid-like fibrils
(Classen et al. 2003 Genes Dev 1714-1726; Elliot et al. 2003, Genes
Dev. 17, 1727-1740).
Hydrophobins are distributed in a water-insoluble form on the
surface of various fungal structures, such as, for example, aerial
hyphae, spores, fruiting bodies. The genes for hydrophobins were
able to be isolated from ascomycetes, deuteromycetes and
basidiomycetes. Some fungi comprise more than one hydrophobin gene,
e.g. Schizophyllum commune, Coprinus cinereus, Aspergillus
nidulans. Different hydrophobins are evidently involved in
different stages of fungal development. The hydrophobins here are
presumably responsible for different functions (van Wetter et al.,
2000, Mol. Microbiol., 36, 201-210; Kershaw et al. 1998, Fungal
Genet. Biol, 1998, 23, 18-33).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: protein gel Example 1.
FIG. 2: protein gel Example 2.
FIG. 3: protein gel Example 6.
FIG. 4: protein gel Example 7.
FIG. 5: protein gel Example 8.
FIG. 6: protein gel Example 9.
DESCRIPTION OF THE INVENTION
Particularly highly suitable hydrophobins for the method according
to the invention are polypeptides of the general structural formula
(I)
X.sub.n--C.sup.1--X.sub.1-50--C.sup.2--X.sub.0-5--C.sup.3--X.sub.p--C.sup-
.4--X.sub.1-100--C.sup.5--X.sub.1-50--C.sup.6--X.sub.0-5--C.sup.7--X.sub.1-
-50--C.sup.8--X.sub.m (I) where X can be any of the 20 naturally
occurring amino acids (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gln,
Arg, Ile Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, Gly) and the
indices alongside X represent the number of amino acids, where the
indices n and m are numbers between 0 and 500, preferably between
15 and 300, p is a number between 1 and 250, preferably 1-100, and
C is cysteine, alanine, serine, glycine, methionine or threonine,
where at least four of the radicals designated C are cysteine, with
the proviso that at least one of the peptide sequences abbreviated
to X.sub.n or X.sub.m or X.sub.p is a peptide sequence at least 20
amino acids in length, which is naturally not linked to a
hydrophobin, which, after coating a glass surface, effect a change
in the contact angle of at least 20.degree..
The amino acids designated C.sup.1 to C.sup.8 are preferably
cysteines; however, they can also be replaced by other amino acids
of similar spatial filling, preferably by alanine, serine,
threonine, methionine or glycine. However, at least four,
preferably at least 5, particularly preferably at least 6 and in
particular at least 7, of the positions C.sup.1 to C.sup.8 should
consist of cysteines. Cysteines can either be present in reduced
form in the proteins according to the invention, or form disulfide
bridges with one another. Particular preference is given to the
intramolecular formation of C--C bridges, in particular those with
at least one, preferably 2, particularly preferably 3 and very
particularly preferably 4, intramolecular disulfide bridges. In the
case of the above-described exchange of cysteines for amino acids
of similar spatial filling, those C positions are advantageously
exchanged in pairs which can form intramolecular disulfide bridges
with one another.
If, in the positions referred to as X, cysteines, serines,
alanines, glycines, methionines or threonines are also used, the
numbering of the individual C positions in the general formulae can
change accordingly.
Particularly advantageous polypeptides are those of the general
formula (II)
X.sub.n--C.sup.1--X.sub.3-25--C.sup.2--X.sub.0-2--C.sup.3--X.sub.5-5-
0--C.sup.4--X.sub.2-35--C.sup.5--X.sub.2-15--C.sup.6--X.sub.0-2--C.sup.7---
X.sub.3-35--C.sup.8--X.sub.m (II) where X can be any of the 20
naturally occurring amino acids (Phe, Leu, Ser, Tyr, Cys, Trp, Pro,
His, Gln, Arg, Ile Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, Gly) and
the indices alongside X represent the number of amino acids, where
the indices n and m are numbers between 2 and 300 and C is
cysteine, alanine, serine, glycine, methionine or threonine, where
at least four of the radicals designated C are cysteine, with the
proviso that at least one of the peptide sequences abbreviated to
X.sub.n or X.sub.m is a peptide sequence which is at least 35 amino
acids in length, which is naturally not linked to a hydrophobin,
which, after coating a glass surface, effect a change in the
contact angle of at least 20.degree..
The origin of the hydrophobins plays no role here. For example, the
hydrophobins can have been isolated, for example, from
microorganisms such as, for example bacteria, yeasts and fungi.
Particularly hydrophobins which have been obtained by means of
genetically modified organisms are suitable according to the
invention.
Suitable additives which can be added to the aqueous hydrophobin
solution are customary, preferably physiologically acceptable
materials.
These include polysaccharides, such as cellulose compounds, pectins
and starches of varying origin, film-forming colloids, such as
gelatin, casein or albumin, mono- or disaccharides, such as
glucose, fructose, lactose or sucrose, or vegetable products, such
as, for example, wheat grit bran or soybean flour.
Furthermore, additives which can be used are inorganic materials,
such as calcium carbonate, clay earths, various forms of
precipitated or mineral silicas and silicates, but also products of
animal origin, such as, for example, eggshell flour. Moreover,
further additives such as emulsifiers, antioxidants or
preservatives can also be used.
The amount of additives used is usually 5 to 200% by weight,
preferably 20 to 200% by weight, based on hydrophobin solid.
The method according to the invention can be carried out as
follows:
The spray auxiliary is introduced together with air or an inert gas
by spraying into a spray device, preferably a spray tower. The
introduction of the spray auxiliary expediently takes place above
the atomization unit. Suitable spray towers are all configurations
known to the person skilled in the art (see e.g. K. Masters, Spray
Drying Handbook, ISBN 0-582-06266-7).
The aqueous hydrophobin solution can be introduced under pressure
through nozzles into the spray space laden with spray auxiliary.
However, it is also possible to allow the aqueous hydrophobin
solution to flow onto rapidly rotating atomizing discs. The design
of the atomization unit has no decisive influence on the product.
Further atomization devices known to the person skilled in the art
can also be used (see e.g. Arthur H. Levebvre, Atomization and
Sprays, ISBN 0-89116-603-3).
The spray cone which forms comprises a large number of small
droplets which are then converted to a dry hydrophobin preparation
by withdrawing water. The drying expediently takes place
immediately after the spraying. Of suitability for this purpose is
evaporative drying, during which the water is removed from the
droplets with the help of a preheated stream of air or inert
gas.
For the drying, particular preference is given to using a fluidized
bed which is located below the atomization unit or in the region of
the atomization unit. The fluidized bed can either be operated as
an integral part of a spray tower (e.g. FSD Technology from Niro or
SBD Technology from Anhydro), or a drying technology is used which
is known to the person skilled in the art as fluidized-bed spray
granulation and is explained, for example, by Hans Uhlemann and
Lothar Morl in the book "Wirbelschicht-Spruhgranulation
[Fluidized-bed spray granulation]", ISBN 3-540-66985-x.
For particularly oxidation-sensitive hydrophobins, the use of inert
gas, such as, for example, nitrogen, during the spraying and the
drying is preferred.
The hydrophobin preparations prepared using this method are
characterized by their good stability and by their low residual
moisture content. The residual moisture is less than 10%, based on
solid substance, measured by Karl-Fischer tritration, preferably,
it is less than 7%, particular preference being given to residual
moisture contents of less than 5%.
The average particle size, measured by means of laser diffraction,
is in the range from 10 micrometers to 3 mm, preference being given
to average particle sizes in the range from 100 micrometers to 1
mm. In particular, the fraction of particles with a size of less
than 50 micrometers should be restricted since this fine fraction
has a tendency toward undesired dust formation during handling.
Particular preference is given to solids with a fraction of
particles measuring less than 50 micrometers of less than 5 mass
%.
The bulk-density of the dry hydrophobin preparation is in the range
from 50 to 1200 kg/m.sup.3. For spray-drying methods in the spray
tower, bulk densities of from 80 to 400 kg/m.sup.3 are preferred,
particularly preferably from 100 to 300 kg/m.sup.3. During the
fluidized-bed spray granulation, bulk densities of from 500 to 1000
kg/m.sup.3 are preferred, particularly preferably from 600 to 800
kg/m.sup.3.
The temperature of the solution to be atomized should usually be 0
to 150.degree. C. In the case of hydrophobins which are easily
inactivated by heat, preference is given to using temperatures of
0-80.degree. C., and in the case of heat-stable hydrophobins, a
temperature of from 20 to 100.degree. C. is preferably used.
Suitable spray auxiliaries are sugar alcohols. Particularly
suitable sugar alcohols are sorbitol, mannitol and inositol.
Furthermore, cellulose, starch, and corn starch are suitable as
drying auxiliary.
The weight amount of spray auxiliary is 5 to 200% by weight,
preferably 20 to 200% by weight, and particularly preferably 70 to
130% by weight, based on hydrophobin solid.
The direct introduction of the spray auxiliary into the spray zone
largely avoids the mechanical stressing of the particles which
results, for example, from a carrier-filled, fluidized bed.
The hydrophobin preparations according to the invention can also be
prepared successfully without using spray auxiliaries.
The novel method is described in detail in the examples below.
General
TABLE-US-00001 Names Hydrophobin A: YaaD-DewA-His6 Hydrophobin B:
40 AA YaaD-DewA-His6 Dry substance content: DS [% by wt.]
Activity Test
To assess the protein activity, the coating properties of the
redissolved spray-dried or spray-granulated hydrophobin fusion
protein are used. The evaluation of the coating properties is
preferably undertaken on glass or Teflon as models for hydrophilic
or hydrophobic surfaces, respectively.
Standard Experiments for Coating
Glass:
concentration of hydrophobin: 50 mg/l incubation of glass plates
overnight (temperature: 80.degree. C.) in 10 mM Tris pH 8 after
coating, washing in demin. water then incubation 10 min/80.degree.
C./1% SDS washing in demin. water Teflon: concentration: 50 mg/l
incubation of Teflon plates overnight (temperature: 80.degree. C.)
in 10 mM Tris pH 8 after coating, washing in demin. water
incubation 10 min/80.degree. C./0.1% Tween 20 washing in demin.
water then incubation 10 min/80.degree. C./1% SDS washing in demin.
water
The samples were dried in the air and the contact angle (in
degrees) of a drop of 5 .mu.l of water was determined. The
following values, for example, were produced:
Mixture with -YaaD-DewA fusion protein (control: without protein;
-:YaaD-DewA-His.sub.6 100 mg/l of purified fusion partner):
TABLE-US-00002 After 1% SDS 80.degree. C. Teflon Glass Control 96.8
30 YaaD 97.4 38.7 50 mg/l 85.9 77.9
Fermentation and Work-Up
In a 1000 ml Erlenmeyer flask with two side chicanes, 200 ml of
complex medium are inoculated with an E. coli strain expressing
YaaD-DewA-His6 or 40 AA YaaD-DewA-H is 6 from LB-Amp plate (100
.mu.g/ml ampicillin) (=first preculture). The strain is incubated
up to an OD.sub.600 nm of ca. 3.5 at 37.degree. C. on a shaker with
d.sub.o=2.5 cm at 200 rpm. Afterwards 4 further 1000 ml Erlenmeyer
flasks with chicanes (in each case with 200 ml of complex medium)
are each inoculated with 1 ml of the first preculture and incubated
at 37.degree. C. in the shaking cabinet (d.sub.o=2.5 cm, n=200 rpm)
(=preculture). As soon as the OD.sub.600 nm is >6, the
prefermenter filled with complex medium is inoculated from this
second shake culture. After reaching an OD.sub.600 nm of >9 or
OTR=80 mmol/(lh), the main fermenter is inoculated. The main
culture is run in the fed-batch method in mineral medium with very
small amounts of complex constituents. At an OD.sub.600 nm of
>70, the cells are induced with 50 .mu.m of IPTG. After an
induction time of between 4 and 20 h, the fermentation is
terminated and the vessel contents are cooled to 4.degree. C. After
the fermentation, the cells are separated off from the fermentation
broth, for example by means of a plate separator (e.g. nozzle
separator) or by microfiltration, and resuspended in demin. water.
Following separation once more by using a plate separator or a
microfiltration, the again resuspended cells are disrupted using a
high-pressure homogenizer at a differential pressure of 2000 bar.
The homogenizate is separated off using a plate separator (e.g.
nozzle separator) and washed several times. The resulting
concentrate is adjusted to pH 12.5. After ca. 15 min, the pH is
lowered to 9. The neutralized hydrophobin-containing solution is
run over a tubular centrifuge to separate off solids. According to
SDS-PAGE analyses, the hydrophobin is present in the supernatant
after the final centrifugation. This supernatant is referred to
below as "aqueous hydrophobin solution". The dry substance content
of the aqueous hydrophobin solution is usually 2-4% by weight. The
hydrophobin concentration determined by means of ELISA is at this
stage typically in the range 4-35 g/l.
Example 1
28.6 kg of mannitol are stirred into 866 kg of aqueous hydrophobin
A solution with a solids content of 3.4% by weight. The solution is
sprayed co-currently into 1200 kg/h of nitrogen using a Gerig Gr.0
type twin-material nozzle at a spraying rate of 41 kg/h. The spray
tower has a diameter of 800 mm and a height of 12 m. The inlet
temperature of the drying gas here is 161 degrees. The exit
temperature of the drying gas is 80 degrees. The deposition takes
place in the filter, in which 31.7 kg of dry material are
retrieved. 6.1 kg of dry material are swept out of the tower. The
contact angles of the redissolved hydrophobin-containing dry
material resulting from the activity test are listed in Table 1.
The protein gel of the redissolved dry material is shown in FIG.
1.
Example 2
2.4 kg of mannitol are stirred into 109 kg of aqueous hydrophobin B
solution with a solids content of 2.4% by weight and a hydrophobin
concentration of 4.5 g/l. The solution is sprayed co-currently into
450 kg/h of nitrogen using a 3 mm Niro twin-material nozzle at a
spraying rate of 12.8 kg/h. The spray tower (manufacturer Niro) has
a diameter of 1200 mm and a cylindrical height of 2650 mm. The
height of the conical section is 600 mm. The inlet temperature of
the drying gas is 163 degrees. The outlet temperature of the drying
gas is 79 degrees. 4.1 kg of dry material are retrieved in the
cyclone discharge. In the downstream filter discharge, 0.73 kg of
dry material are retrieved. The contact angles of the redissolved
hydrophobin-containing dry material resulting from the activity
test are listed in Table 2. The protein gel of the redissolved dry
material is shown in FIG. 2.
Example 3
5.4 kg of sodium sulfate are stirred into 155 kg of aqueous
hydrophobin A solution with a solids content of 3.4% by weight and
a hydrophobin content of 31 g/l. The solution is sprayed
co-currently into 450 kg/h of nitrogen at a spraying rate of 12.2
kg/h via a 3 mm Niro twin-material nozzle. The spray tower
(manufacturer NIRO) has a diameter of 1200 mm and a cylindrical
height of 2650 mm. The height of the conical section is 600 mm. The
inlet temperature of the drying gas here is 165 degrees. The outlet
temperature of the drying gas is 84 degrees. In the cyclone
discharge, 5.5 kg of dry material are retrieved.
Example 4
40 g of sodium sulfate are stirred into 2 l of aqueous hydrophobin
B solution with a dry substance content of 20 g/l. The solution is
dried, in a Buchi laboratory spray-dryer with nitrogen as drying
gas. The gas inlet temperature is 160 degrees. The gas outlet
temperature is 80.degree. C. In the cyclone discharge, 40 g are
retrieved. The resulting contact angles of the redissolved dry
material are listed in Table 3.
Example 5
40 g of maltodextrin are stirred into 2 l of aqueous hydrophobin B
solution with a dry substance content of 20 g/l. The solution is
dried in a Buchi laboratory spray-dryer with nitrogen as drying
gas. The gas inlet temperature is 160 degrees. The gas outlet
temperature is 80.degree. C. In the cyclone discharge, 39 g are
retrieved. The resulting contact angles of the redissolved dry
material are listed in Table 4.
Example 6
240 kg of aqueous hydrophobin A solution (TS=3.4% by weight,
hydrophobin concentration 31 g/l) are concentrated. This results in
65 kg of aqueous hydrophobin A solution with a solids content of
13% by weight and a hydrophobin content of 117 g/l. This
concentrated solution is sprayed into 450 kg/h of nitrogen at a
spraying rate of 13.7 kg/h via a 3 mm Niro twin-material nozzle.
The spray tower (manufacturer NIRO) has a diameter of 1200 mm and a
cylindrical height of 2650 mm. The height of the conical section is
600 mm. The inlet temperature of the drying gas here is 165
degrees. The outlet temperature of the drying gas is 84 degrees. In
the cyclone discharge, 6.1 kg of dry material are retrieved. The
contact angles of the redissolved hydrophobin-containing dry
material resulting from the activity test are listed in Table 5.
The protein gel of the redissolved dry material is shown in FIG.
3.
Example 7
894 kg of aqueous hydrophobin B solution (TS=2.4% by weight,
hydrophobin concentration 6.3 g/l) are concentrated. This results
in 226 kg of concentrated aqueous hydrophobin B solution (TS=9.7%
by weight, hydrophobin concentration 35.4 g/l). 75 kg of this
concentrated aqueous hydrophobin B solution with a solids content
of 9.6% (hydrophobin content ca. 35.4 g/l) is sprayed co-currently
into 450 kg/h of nitrogen at a temperature of 25 degrees using a
single-material nozzle (Niro) with a diameter of 2 mm at a spraying
rate of 13.6 kg/h. The spray tower (manufacturer NIRO) has a
diameter of 1200 mm and a cylindrical height of 2650 mm. The height
of the conical section is 600 mm. The inlet temperature of the
drying gas here is 168 degrees. The exit temperature of the drying
gas is 84 degrees. In the cyclone, 5 kg of product were retrieved,
and in the filter 1.2 kg of product were retrieved. The spray-dried
material has a bulk density of 100 kg/m.sup.3. The purity of the
resulting material, which is defined as a percentage of hydrophobin
to total protein concentration, is 45%. The contact angles of the
redissolved hydrophobin-containing dry material resulting from the
activity test are listed in Table 6. The protein gel of the
redissolved dry material is shown in FIG. 4.
Example 8
921 kg of aqueous hydrophobin A solution (TS=2.3% by weight,
hydrophobin concentration of 18 g/l) are concentrated. This results
in 241 kg of concentrated aqueous hydrophobin A solution (TS=9.6%
by weight, hydrophobin concentration 98.3 g/l). This concentrated
aqueous hydrophobin A solution is introduced into a spray fluidized
bed (d=150 mm, A=0.177 m.sup.2) with initial charge of dried
hydrophobin A. The spraying of the concentrated aqueous hydrophobin
A solution takes place via a 2-material nozzle with a diameter of 2
mm.
Part 1: 2 kg of hydrophobin A from Example 4 are initially
introduced. The spraying rate of the concentrated aqueous
hydrophobin A solution (=feed 1) is increased in the steps 1.8
kg/h; 2.5 kg/h; 3.3 kg/h. The gas inlet temperature at a product
spraying rate of 3.3 kg/h is 138 degrees and the associated drying
gas stream (air) is 75 m.sup.3/h. The average product outlet
temperature in the lower region of the spray fluidized bed is 69
degrees. The granules are continuously discharged via a discharge
screw. The particle size is controlled via the screening of the
discharge and appropriate grinding of the coarse material in a
grain cutter. From 5.9 kg of feed 1, 0.44 kg of dry material result
(sample 1, FIG. 5).
Part 2: concentrated aqueous hydrophobin A solution (=feed 2) is
sprayed onto the dry material remaining in the fluidized bed from
part 1 (1.5 kg, particle size of the initial charge <1.25 mm) at
a spraying rate of 3.2 kg/h. The average gas inlet temperature is
126 degrees and the associated stream of drying gas (air) is 102
m.sup.3/h. The average product outlet temperature in the lower
region of the spray fluidized bed is 67 degrees. The granules are
continuously discharged via a discharge screw. The particle size is
controlled via the screening of the discharge and appropriate
grinding of the coarse material in a grain cutter. From 21.3 kg of
feed 2, 1.6 kg of dry material result (sample 2, FIG. 5).
Part 3: concentrated aqueous hydrophobin A solution (=feed 3) is
sprayed onto the dry material remaining in the fluidized bed from
part 2 (1.3 kg) at a spraying rate of 3.6 kg/h. The average gas
inlet temperature is 143 degrees and the associated stream of
drying gas (air) is 102 m.sup.3/h. The average product outlet
temperature in the lower region of the spray fluidized bed is 67
degrees. The granules are discharged continuously via a discharge
screw. The particle size is controlled via the screening of the
discharge and appropriate grinding of the coarse material in a
grain cutter. From 21.6 kg of feed 3, 1.6 kg of dry material result
(sample 3, FIG. 5).
Part 4: concentrated aqueous hydrophobin A solution (=feed 4) is
sprayed onto the dry material from part 3 remaining in the
fluidized bed (1.9 kg, particle size of the initial charge <1.6
mm) at a spraying rate of 4.1 kg/h. The average gas inlet
temperature is 147 degrees and the associated stream of drying gas
(air) is 100 m.sup.3/h. The average product outlet temperature in
the lower region of the spray fluidized bed is 66 degrees. The
granules are continuously discharged via a discharge screw. The
particle size is controlled via the screening of the discharge and
appropriate grinding of the coarse material in a grain cutter. From
24.6 kg of feed 4, 1.9 kg of dry material result (sample 4, FIG.
5).
Part 5: concentrated aqueous hydrophobin A solution (=feed 5) is
sprayed onto the dry material from part 4 remaining in the
fluidized bed (1.3 kg, particle size of the initial charge <1.4
mm) at a spray rate of 4 kg/h. The average gas inlet temperature is
146 degrees and the associated stream of drying gas (air) is 99
m.sup.3/h. The average product outlet temperature in the lower
region of the spray fluidized bed is 67 degrees. The granules are
continuously discharged via a discharge screw. The particle size is
controlled via the screening of the discharge and appropriate
grinding of the coarse material in a grain cutter. From 23 kg of
feed 5, 1.7 kg of dry material result (sample 5, FIG. 5).
Part 6: concentrated aqueous hydrophobin A solution (=feed 6) is
sprayed onto the dry material from part 5 remaining in the
fluidized bed (1.1 kg, particle size of the initial charge <1.25
mm) at a spraying rate of 3.5 kg/h. The average gas inlet
temperature is 142 degrees and the associated stream of drying gas
(air) is 101 m.sup.3/h. The average product outlet temperature in
the lower region of the spray fluidized bed is 71 degrees. The
granules are continuously discharged via a discharge screw. The
particle size is controlled via the screening of the discharge and
appropriate grinding of the coarse material in a grain cutter. From
26.8 kg of feed 6, 2 kg of dry material result (sample 6, FIG. 5).
The bulk density of the useful fraction is 0.65 kg/I.
Part 7: concentrated aqueous hydrophobin A solution (=feed 7) is
sprayed onto the dry material from part 6 remaining in the
fluidized bed (1.2 kg, particle size of the initial charge <1.25
mm) at a spraying rate of 3.7 kg/h. The average gas inlet
temperature is 141 degrees and the associated stream of drying gas
(air) is 99 m.sup.3/h. The average product outlet temperature in
the lower region of the spray fluidized bed is 72 degrees. The
granules are continuously discharged via a discharge screw. The
particle size is controlled via the screening of the discharge and
appropriate grinding of the coarse material in a grain cutter. From
22.7 kg of feed 7, 1.7 kg of dry material result (sample 7, FIG.
5). The bulk density of the useful fraction is 0.65 kg/l. The
fraction between 0.2-0.72 mm particle size is 98.9%.
Besides using the grain cutter in this example, a roller mill or a
grinder or the like is suitable for grinding the coarse
material.
The contact angles of the redissolved hydrophobin-containing dry
material resulting from the activity test are listed in Tab. 7. The
protein gel of the redissolved dry material is shown in FIG. 5.
Example 9
894 kg of aqueous hydrophobin B solution (DS=2.4% by weight,
hydrophobin concentration 6.3 g/l) are concentrated. 226 kg of
concentrated aqueous hydrophobin B solution result (DS=9.7% by
weight, hydrophobin concentration 35.4 g/l).
Part 1: 1 kg of the hydrophobin B-containing dry material from a
spray drying analogous to Example 5 is initially introduced. The
gas inlet temperature is 141 degrees and the associated stream of
drying gas (air) is 49 m.sup.3/h. The average product outlet
temperature in the lower region of the spray fluidized bed is 69
degrees. The granules are continuously discharged via a discharge
screw. The particle size is controlled via the screening of the
discharge and appropriate grinding of the coarse material in a
grain cutter. From 5 kg of feed 1, 0.46 kg of dry material
result.
Part 2: concentrated aqueous hydrophobin B solution (=feed 2) is
sprayed onto the dry material from part 1 remaining in the
fluidized bed (0.87 kg; particle size <0.8 mm) at a spraying
rate of 3.8 kg/h. The gas inlet temperature is 139 degrees and the
associated stream of drying gas (air) is 99 m.sup.3/h. The average
product outlet temperature in the lower region of the spray
fluidized bed is 67 degrees. The granules are continuously
discharged via a discharge screw. The particle size is controlled
via the screening of the discharge and appropriate grinding of the
coarse material in a grain cutter. From 22.8 kg of feed 2, 2.1 kg
of dry material result. The fraction <0.4 mm is ca. 80%.
Part 3: concentrated aqueous hydrophobin B solution (=feed 3) is
sprayed onto the dry material from part 2 remaining in the
fluidized bed (0.87 kg, particle size <0.8 mm) at a spraying
rate of 3.8 kg/h. The gas inlet temperature is 140 degrees and the
associated stream of drying gas (air) is 102 m.sup.3/h. The average
product outlet temperature in the lower region of the spray
fluidized bed is 69 degrees. The granules are continuously
discharged via a discharge screw. The particle size is controlled
via the screening of the discharge and appropriate grinding of the
coarse material in a grain cutter. From 30.1 kg of feed 3, 2.78 kg
of dry material result. The fraction between 0.4-0.8 mm is 80%.
Part 4: concentrated aqueous hydrophobin B solution (=feed 4) is
sprayed onto the dry material from part 3 remaining in the
fluidized bed (1.1 kg, particle size <0.8 mm) at a spraying rate
of 3.5 kg/h. The gas inlet temperature is 142 degrees and the
associated stream of drying gas (air) is 96 m.sup.3/h. The average
product outlet temperature in the lower region of the spray
fluidized bed is 72 degrees. The granules are continuously
discharged via a discharge screw. The particle size is controlled
via the screening of the discharge and corresponding grinding of
the coarse material in a grain cutter. From 25.9 kg of feed 4, 2.4
kg of dry material result. The fraction from 0.4-0.8 mm comprises
ca. 80%.
Part 5: concentrated aqueous hydrophobin B solution (=feed 5) is
sprayed onto the dry material from part 4 remaining in the
fluidized bed (1.1 kg, particle size <0.8 mm) at a spraying rate
of 3.4 kg/h. The gas inlet temperature is 139 degrees and the
associated stream of drying gas (nitrogen) is 99 m.sup.3/h. The
average product outlet temperature in the lower region of the spray
fluidized bed is 72 degrees. From 26.6 kg of feed 5, 2.5 kg of dry
material result. The granules are continuously discharged via a
discharge screw. The particle size is controlled via the screening
of the discharge and appropriate grinding of the coarse material in
a grain cutter. The fraction from 0.2-0.8 mm comprises ca. 98%.
Part 6: concentrated aqueous hydrophobin B solution (=feed 6) is
sprayed onto the dry material from part 5 remaining in the
fluidized bed (1.1 kg, particle size <0.8 mm) at a spraying rate
of 3.4 kg/h. The gas inlet temperature is 142 degrees and the
associated stream of drying gas (nitrogen) is 98 m.sup.3/h. The
average product outlet temperature in the lower region of the spray
fluidized bed is 72 degrees. From 25.4 kg of feed 6, 2.4 kg of dry
material result. The granules are continuously discharged via a
discharge screw. The particle size is controlled via the screening
of the discharge and appropriate grinding, of the coarse material
in a grain cutter. The fraction from 0.2-0.8 mm comprises 91-95%.
The bulk density of the sample is 0.57 kg/l.
Part 7: concentrated aqueous hydrophobin B solution (=feed 7) is
sprayed onto the dry material from part 6 remaining in the
fluidized bed (1.4 kg, particle size <0.8 mm) at a spraying rate
of 3.4 kg/h. The gas inlet temperature is 140 degrees and the
associated stream of drying gas (nitrogen) is 100 m.sup.3/h. The
average product outlet temperature in the lower region of the spray
fluidized bed is 72 degrees. The granules are continuously
discharged via a discharge screw. The particle size is controlled
via the screening of the discharge, and appropriate grinding of the
coarse material in a grain cutter. From 18.4 kg of feed 7, 1.8 kg
of dry material result. The fraction from 0.2-0.4 mm is >0.64
kg/l.
Part 8: concentrated aqueous hydrophobin B solution (=feed 8) is
sprayed onto the dry material from part 7 remaining in the
fluidized bed (1.2 kg, particle size <0.8 mm) at a spraying rate
between 3 and 2.5 kg/h. The gas inlet temperature is 120 degrees
and the associated stream of drying gas (nitrogen) is 100
m.sup.3/h. The average product outlet temperature in the lower
region of the spray fluidized bed is 67 degrees. The granules are
continuously discharged via a discharge screw. The particle size is
controlled via the screening of the discharge and appropriate
grinding of the coarse material in a grain cutter. From 21.5 kg of
feed 8, 2.1 kg of dry material result (FIG. 6). The proportion of
the fraction between 0.2-0.4 mm is >70.5%.
Part 9: concentrated aqueous hydrophobin B solution (=feed 3) is
sprayed onto the dry material from part 8 remaining in the
fluidized bed (1.2 kg, particle size <0.8 mm) at a spraying rate
of 4.1-4.4 kg/h. The gas inlet temperature is 163 degrees and the
associated stream of drying gas (nitrogen) is 97 m.sup.3/h. The
average product outlet temperature in the lower region of the spray
fluidized bed is 77 degrees. The granules are continuously
discharged via a discharge screw. The particle size is controlled
via the screening of the discharge and appropriate grinding of the
coarse material in a grain cutter. From 29.6 kg of feed 9, 2.8 kg
of dry material result. The proportion of the fraction between
0.2-0.4 mm is 14.8%.
Part 10: 1.3 kg of the coarse material ground under part 9 are
initially introduced together with the fines (particle size of the
initial charge <0.8 mm). The spraying rate of the concentrated
aqueous hydrophobin B solution (=feed 10) is varied between 4.9 and
5.1 kg/h. The gas inlet temperature is 181 degrees and the
associated stream of drying gas (nitrogen) is 95 m.sup.3/h. The
average product outlet temperature in the lower region of the spray
fluidized bed is 82 degrees. The granules are continuously
discharged via a discharge screw. The particle size is controlled
via the screening of the discharge and appropriate grinding of the
coarse material in a grain cutter. From 43.1 kg of feed 10, 4.1 kg
of dry material result (sample 10, FIG. 6). The proportion of the
fraction between 0.2-0.4 mm is 12.6%.
The contact angles of the redissolved hydrophobin-containing dry
material of the end sample (part 10) resulting from the activity
test are listed in Table 8. The protein gel of the redissolved dry
material is shown in FIG. 6.
TABLE-US-00003 TABLE 1 Contact angles after spray drying of
hydrophobin A with mannitol. Glass Teflon Control 20.5 108.2
Example 1 66.2 85.5
TABLE-US-00004 TABLE 2 Contact angles after spray drying of
hydrophobin B with mannitol. Glass Teflon Control 21.1 108.6
Example 2 68.3 78.2
TABLE-US-00005 TABLE 3 Contact angles after spray drying of
hydrophobin A with sodium sulfate. Glass Teflon Control 15 110.8
Example 4 64.9 86.3
TABLE-US-00006 TABLE 4 Contact angles after spray drying of
hydrophobin A with maltodextrin. Glass Teflon Control 15 110.8
Example 5 65.7 85.7
TABLE-US-00007 TABLE 5 Contact angles after spray drying of
hydrophobin A without auxiliary. Glass Teflon Control 15.5 107
Example 6 65.9 85.6
TABLE-US-00008 TABLE 6 Contact angles after spray drying of
hydrophobin B without auxiliary. Glass Teflon Control 32.8 96.2
Example 7 69.6 74.8
TABLE-US-00009 TABLE 7 Contact angles after spray granulation of
hydrophobin A without auxiliary. Example 8 Glass Teflon Control 13
97.8 Part 1 60.7 75.9 Part 2 62.2 81.2 Part 3 60.5 81.9 Part 4 60.9
62.5 Part 5 58.9 70.1 Part 6 60 72.5 Part 7 59.3 72.9
TABLE-US-00010 TABLE 8 Contact angles after spray granulation of
hydrophobin B without auxiliary. Glass Teflon Control 23.3 100.8
Example 9, 72.1 69.7 end sample, Part 10
EXPLANATION OF THE FIGURES
FIG. 1: protein gel Example 1:
4-12% Bis-Tris gel/MES buffer, left: after spray drying of
hydrophobin A with mannitol, right: marker: prestained SDS-Page
standards, application/slot: 15 .mu.g Pr,
FIG. 2: protein gel Example 2:
FIG. 3: protein gel Example 6:
4-12% Bis-Tris gel/MES buffer, left: marker: prestained SDS-Page
standards, application/slot: 15 .mu.g Pr, right: after spray drying
of hydrophobin A without auxiliary.
FIG. 4: protein gel Example 7:
4-12% Bis-Tris gel/MES buffer, left: marker: prestained SDS-Page
standards, application/slot: 15 .mu.g Pr, center/right: after spray
drying of hydrophobin B without auxiliary.
FIG. 5: protein gel Example 8:
4-12% Bis-Tris gel/MES buffer, left: marker: prestained SDS-Page
standards, application/slot: 15 .mu.g Pr, remainder: parDS 1-7
after spray granulation of hydrophobin A without auxiliary.
FIG. 6: protein gel Example 9:
4-12% Bis-Tris gel/MES buffer, left: marker: prestained SDS-Page
standards, application/slot: 15 .mu.g Pr, right: sample 10 after
spray granulation of hydrophobin B without auxiliary.
* * * * *